Abstract

Background

Trehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar,
carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose
6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be
a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic
plant-microbe interactions, in responses to abiotic stress and in developmental signalling,
but its precise functions are unknown. A major obstacle to investigating its function
is the technical difficulty of measuring the very low levels of trehalose usually
found in plant tissues, as most of the established trehalose assays lack sufficient
specificity and/or sensitivity.

Results

A kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose.
Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose
content of the tissue extract is determined from an internal calibration curve. The
assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ≥88%.
A. thaliana Col-0 rosettes contain about 20–30 nmol g-1FW of trehalose, increasing to about 50–60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas
a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents
of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively.

Conclusion

The fluorometric assay is highly specific for trehalose and sensitive enough to measure
the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold
accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution
to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status.
The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia
or a downward shift in the trehalose:Tre6P ratio.